Learning and memory are likely to involve changes in the efficacy of synaptic transmission. In many cases, these changes are thought to involve modulation of the mechanism by which transmitter is released from the nerve terminal. This proposal attempts to illuminate some of the fundamental cellular mechanisms by which synapses release transmitter, on the presumption that understanding these mechanisms is a precondition to fully understanding how release is altered by learning or by disease states. The approach of this proposal is to use Drosophila genetics to explore two proteins appear to be central to this process: synaptotagmin and synaprobrevin. Both proteins are on synaptic vesicles and Drosophila mutations have been isolated in both genes. Mutations will be characterized by a combination of electrophysiology, electron microscopy, and the use of a fluorescent dye to monitor membrane trafficking. To probe the actions of these proteins more closely, engineered alteration in these proteins will be reintroduced into the mutant files. Drosophila will also be used to screen for novel mutations that may reveal previously unknown components of the synaptic machinery. Specifically, this project will: 1. Create alterations in particular domains of synaptotagmin for biochemical and genetic experiments. 2. By examining the phenotype of these altered synaptotagmins to test the hypotheses that synaptotagmin is the Ca2+ -sensor for triggering vesicle fusion, a regulator of vesicle docking, and involved in vesicle recycling. 3. Use Drosophila that lack synaprobrevin and use cloned genes encoding additional members of the v-SNARE family to identify sequences essential for targeting synaptobrevin to vesicles and targeting vesicles to their appropriate release sites. 4. Use altered synaptobrevins to determining which regions are essential for vesicle fusion with the plasma membrane. 5. Screen for enhancers and suppressors of known synaptic mutations and characterize those mutations. 6. Screen physiologically for mutations in synaptic transmission and characterize those mutations.